pfizer-confidential-translated

Masking area: under adjustment

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

TABLE OF CONTENTS
ES TO LE acnsoasavadtvethniatatsda cnseiiassscapraadbeansscadiniasinseanaaasa std eaatlebsinetannianaareinatereanaiiiels 1
Lilies we st Pale a ceca chest catia sca Meamaanmsauaesaaniehca thane dionia eau baganes tetas aeie een den bee 1
Terms and abbreviations used in this S@CtION........cccecsessesssesceeseeceecaeseeesaesaeaesaneeeeeeeeeaaeeeeeneaes 2
OU SIMA ics tecectascsdeeecns tee 2tsceesccer tach cee iesases asf: caeeoecns jena lbscts iaceeiacesaccanecacemecatmed aces lath aletea sues ecassogesite 2
CA LN GIS IS ASI cea ghclid a (610 meerertee terre eter tere tree ceeepeer aceer cere errr ert feet eeeeere rere errr et een tee ee Bee orr reer eee 4
SO SOI OURO Pica 2ecenanex Goes svenrstvewlees erat yiirine retniog Wee eecar toes tam arene oneeteninnee bares aed 4
NaS idle vite tet eter err retraite ent tener ret err tre te 5
Mis ITI UA Se sata este ae ce a acy aa tear a a bees eee ewes 7
BE MOPS UI Wiss says onus aaa aooansnamenduansaasaaasaush cuonshauantaddavenesst Gantt saanancaniaays ioda: iaamiauaaias ania eae anaes 9
1 PRATMACGRINGEIC LKUE Meera CTOs) x ztc2e¢cctecsecstaretie atazenctsavietaateneettian neve merece ace’ 9
o Other FialiniaGa cite: We SiS. ss1is125 147 cassiexsviareriacy acd tein eeteden tics dain ctniesstaiecbetes 9
9 ., DISCUSS foliaitid COMGHISIOWsissscsesccsecsicessscosiedecnaabed aedsectacsesed sasessceeansgetdedicbateeceastsagieneaed ooqeacd 9
DOE TE VS US se ccteags sca nc sane sac odes mats beats syaai acne ondaciadanesaneeswats aaatieap aia Taiud ean bal ee eaten 10
PP GN ante cee tas cece oe ae cease cece eae soap oven nce aces oz aves ese neat cent oeeeses ees 10
LIST OF TABLES

Table 1 Pharmacokinetics of luciferase RNA-encapsulated LNPs, ALC-0315 and ALC-
0159, when administered intravenously to Wistar Han rats at a dose of 1 mg
FUP crass pccpassdoeas tesa tardtaontpianratosusdsevqnisesiea’ ciutertoyinmnsatnatirsesnas pases iAaaemmaeatneinenerasieh 4
LIST OF FIGURES
Figure 1 Plasma and liver concentrations of ALC-0315 and ALC-0159 after intravenous
administration of luciferase RNA-encapsulated LNPs at a dose of 1 mg RNA/kg to Wistar
MTN lite ss Apaetsestbcoseslster!saacida cetececsttestaeeceleenctenarcanacte vente secebiaat stediatesectisectataceatesin pialusectitaveuersisiacacties 5
Figure 2 In vivo luminescence in BALB/c mice intramuscularly treated with luciferase
BeAr Ce SUIS CLIN IP fences cient cance ta ceaade selec snadsehadeteacai tea ieshoensaeeascicoddaenaaqesacasenanerctauienudsciaumtnccis 6
Figure 3 Estimated in vivo metabolic pathways of ALC-0315 in various animal species..8

Figure 4 Estimated in vivo metabolic pathways of ALC-0159 in various animal species. 9

PFIZER CONFIDENTIAL
Page 1

Masking area: under adjustment

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

Terms and abbreviations used in this section

Terms and abbreviations

Unabridged expressions or definitions

ALC-0159 PEG lipids added to the drug

ALC-0315 Aminolipids added to this product

[sH]-CHE Radiolabeled [Cholesteryl-1,2-3H(N)]-Cholesteryl Hexadecyl Ether

DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine

GLP Good Laboratory Practice

LNP Lipid-nanoparticle

modRNA Nucleoside-modified mRNA

mRNA Messenger RNA

m/z m/z

PEG Polyethylene glycol

PK Pharmacokinetics

RNA Ribonucleic acid

S9 Supernatant fraction obtained from liver homogenate by
centrifuging at 9000 g

WHO World Health Organization

PFIZER CONFIDENTIAL
Page 2

Masking area: under adjustment

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

1. Summary

BNT162b2 (BioNTech code: BNT162, Pfizer code: PF-07302048) is a modified nucleoside
mRNA (modRNA) encoding the full-length spike glycoprotein (S protein) of severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2). It is being developed as the essence
of an MRNA vaccine against infections caused by SARS-CoV-2. To formulate BNT162b2,
it was mixed with two functional lipids, ALC-0315 (aminolipid) and ALC-0159 (PEG-lipid),
and two structural lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and
cholesterol. (1,2-distearoyl-sn-glycero-3-phosphocholine) as two structural lipids, and
cholesterol to form lipid nanoparticles (LNPs) that encapsulate BNT162b2 (henceforth,
"BNT162b2 encapsulated LNPs").

To evaluate the non-clinical pharmacokinetics of BNT162b2-encapsulated LNPs, we
performed evaluation studies both in vivo and in vitro to see the absorption (PK),
metabolism, and excretion of ALC-0315 and ALC-0159 in LNPs, as well as biodistribution
studies using luciferase or radioactively labeled lipid as alternative reporters for
BNT162b2.

Based on the fact that the development of vaccines for the prevention of infectious
diseases does not require evaluation of systemic exposure (WHO, 2005; Guidelines for
Non-clinical Studies of Vaccines for the Prevention of Infectious Diseases) 1,2, we did not
conduct a PK study using intramuscular administration of BNT162b2-encapsulated LNP.
The other two lipids contained in the drug (cholesterol and DSPC) are naturally occurring
lipids and are considered to be metabolized and excreted in the same manner as
endogenous lipids. In addition, it is expected that BNT162b2 is degraded by ribonuclease
in uptake cells and metabolized by nucleic acid, and that BNT162b2-derived S protein
undergoes proteolysis. Based on the above, it was not considered necessary to evaluate

the metabolism and excretion of these components again.

As an alternative reporter to BNT162b2, luciferase RNA-encapsulated LNPs (luciferase

RNA encapsulated in LNPs with the same lipid composition as BNT162b2-encapsulated

LNPs; hereafter referred to as "luciferase RNA-encapsulated LNPs") were intravenously
administered to Wistar Han rats. In the study, plasma, urine, feces, and liver samples
were collected over time, and the concentrations of ALC-0315 and ALC-0159 were
measured in each sample. The results showed that ALC-0315 and ALC-0159 were rapidly
distributed from the blood to the liver. About 1% and 50% of the dose of ALC-0315 and
ALC-0159, respectively, were excreted in the feces as unchanged drug, and both were

below the detection limit in the urine.

In the biodistribution study, luciferase RNA-encapsulated LNP was intramuscularly
administered to BALB/c mice. In the biodistribution study, luciferase RNA-encapsulated
LNP was intramuscularly administered to BALB/c mice, and the expression of luciferase
was observed at the site of administration and also in the liver where the level of
expression was even lower. Expression of luciferase at the site of administration was
observed at 6 hours post-dose and disappeared by 9 days post-dose. Expression of
luciferase in the liver was also observed at 6 hours post-dose and disappeared by 48
hours post-dose. The radioactivity of the radioactively labeled luciferase RNA-
encapsulated LNPs was intramuscularly administered to rats, and the biodistribution of
the radioactivity was quantitatively evaluated. The highest non-dose site was the liver
(up to 18% of dose).

The metabolism of ALC-0315 and ALC-0159 was evaluated in vitro using blood, liver
microsomes, liver S9 fractions and hepatocytes from CD-1/ICR mice, Wistar Han or
Sprague Dawley rats, crab-eating macaques or humans. In vivo metabolism was also
investigated using plasma, urine, feces, and liver samples collected from the intravenous
PK study in rats. These in vitro and in vivo studies showed that ALC-0315 and ALC-0159
were slowly metabolized by hydrolysis of ester and amide bonds, respectively, in all

animal species tested.

PFIZER CONFIDENTIAL
Page 3

Masking area: under adjustment

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

These non-clinical pharmacokinetic evaluations indicated that LNP that reached the
circulation was distributed to the liver. In addition, the disappearance of ALC-0315 and

ALC-0159 was suggested to be related to metabolism and fecal excretion, respectively.

2. Analysis method

Report Number: PF-07302048_06 72424

An LC/MS method with appropriate performance was developed to quantify the
concentrations of ALC-0315 and ALC-0159, the constituent lipids of LNP, in the non-GLP
rat intravenous PK study (Section M2.6.4.3). In other words, 20 wL of plasma, liver
homogenate (homogenates were prepared from sections taken from three different
locations of the liver and pooled and diluted with blank matrix as appropriate), urine and
fecal homogenate (diluted with blank matrix as appropriate) samples were each
deproteinized in acetonitrile containing an internal standard (PEG-2000). The samples

were centrifuged and the supernatant was used for LC-MS/MS measurements.

3. Absorption

Report Number: PF-07302048_06 72424, Summary Table: 2.6.5.3

To investigate the pharmacokinetics of ALC-0315 and ALC-0159, luciferase RNA-
encapsulated LNPs were administered intravenously to male Wistar Han rats at a single
dose of 1 mg RNA/kg, and plasma and liver samples were collected by sparse sampling
over time (0.1, 0.25, 0.5, 1, 3, 6, and 24 h before, and 2, 4, 8, and 14 days after
administration). Plasma and liver samples were collected by sparse sampling (3
animals/time point). Plasma and liver concentrations of ALC-0315 and ALC-0159 were
measured and PK parameters were calculated (Table 1). ALC-0315 and ALC-0159 in
blood were promptly distributed to the liver by 24 hours after administration. Plasma
concentrations of ALC-0315 and ALC-0159 at 24 hours post-dose were less than 1% of
the maximum plasma concentration (Figure 1). The apparent terminal phase elimination
half-lives (t%) were similar in plasma and liver, 6-8 days for ALC-0315 and 2-3 days for
ALC-0159. The results of this study suggest that the liver is one of the major tissues that
take up ALC-0315 and ALC-0159 from the blood.

The results of the investigation of urinary and fecal concentrations of ALC-0315 and ALC-
0159 conducted in this study are described in section M2.6.4.6.

Table 1 Pharmacokinetics of ALC-0315 and ALC-0159 in LNP included with luciferase labeled RNA injected
intravenously in Wistar Han Rat at Img RNA/kg

component Amountofcomponent Sex/N t% (h) AUCint AUCaast Distribution in
(mg/kg) (ugeh/mL) (ug*h/mL) the Liver (%)
ALC-0315 15.3 Male/3> 139 1030 1020 60
ALC-0159 1.96 Male/3> 72.7 99.2 98.6 20

a. Calculated by [Max amount of distribution in the Liver]/[Total amount injected]
b. 3 animals in each point. Spars sampling

PFIZER CONFIDENTIAL
Page 4

Masking area: under adjustment

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

Figure 1 Plasma and liver concentrations of ALC-0315 and ALC-0159 after intravenous

administration of luciferase RNA-encapsulated LNPs at a dose of 1 mg RNA/kg to Wistar
Han rats

=—@— ALC-0315. inplasma : =— @ — ALC-0315| in the Liver
6 = @ = ALC-0159) in the Liver
10% —e— ALC-0159 in plasma is ? O~n g_ se cai
O -_—
b =O --e---- ©
tu)
E D> 10 &
= = \
Cc c a
B=] 2 ‘
£ 01 g *.
c fo ~
8 8 i —
36) fo) ap
O 1S)
001
) 100 200 300 0 100 20 yx
Time after injection (h) Time after injection (h)

4. Distribution

Report No.: REoo72, 185350, Summary Table: 2.6.5.5A, 2.6.5.5B

Luciferase RNA-encapsulated LNPs were administered to three female BALB/c mice,
and the biodistribution of BNT162b2 was examined using luciferase luminescence as a
surrogate marker. In other words, luciferase RNA-encapsulated LNPs_ were
intramuscularly administered to the left and right hind paws of mice at a dose of 1 wg
RNA each (2 wg RNA in total). Luciferin, a luminescent substrate, was then administered
intraperitoneally 5 min before detection of luciferase luminescence, and in vivo
luminescence was measured under isoflurane anesthesia at 6 and 24 hours after
administration and at 2, 3, 6, and 9 days using Xenogen IVIS Spectrum. The expression
of luciferase protein was evaluated over time in the same individual. The expression of
luciferase at the site of administration was observed from 6 hours post-dose and
disappeared by 9 days post-dose. The expression of luciferase in the liver was also

observed from 6 hours post-dose and disappeared by 48 hours post-dose. The

distribution in the liver was considered to indicate that a portion of the locally
administered luciferase RNA-encapsulated LNP reached the circulating blood and was
taken up by the liver. As described in detail in Section M2.6.4.3, when luciferase RNA-
encapsulated LNPs were administered intravenously to rats, the liver was suggested to
be the major organ of distribution for ALC-0315 and ALC-0159, which is consistent with
the findings of this study in which ALC-0315 and ALC-0159 were administered
intramuscularly to mice. No toxicity findings indicating hepatic injury were observed in

the rat repeated-dose toxicity study (Section 2.6.6.3).

PFIZER CONFIDENTIAL
Page 5

Masking area: under adjustment

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

Figure 2 In vivo luminescence in BALB/c mice intramuscularly treated with luciferase

6h

24h

6d

RNA-encapsulated LNP

10*°

10°

¢ +

10°
10’
10°
10° © o—_e——_ °

10

Luciferase RNA-encapsulated LNPs using [3H]-cholesteryl hexadecyl ether ([3H]-CHE)-
labeled LNPs were administered intramuscularly to male and female Wistar Han rats at

a dose of 50 wg RNA, and blood, plasma, and tissues were collected from three animals
per sex at 15 min and at 1, 2, 4, 8, 24, and 48 h after administration. Blood, plasma, and

tissue samples were collected from three animals each at 15 min and 1, 2, 4, 8, 24, and

48 h post-dose, and the biodistribution of LNPs was evaluated by measuring the

radioactivity concentration by liquid scintillation counting. In both males and females, the

highest radioactivity levels were observed at the site of administration at all
measurement time points. Plasma radioactivity levels were highest in the first 1 to 4
hours post-dose. The highest radioactivity levels in these tissues were observed 8 to 48
hours post-dose. Total recoveries of radioactivity relative to the dose outside of the dose
site were highest in the liver (up to 18%) and were significantly lower in the spleen
(<1.0%), adrenal glands (<0.11%), and ovaries (<0.095%) than in the liver. The mean
radioactivity concentrations and tissue distribution patterns were generally similar in

males and females.

The distribution of the antigen encoded by BNT162b2 in vivo is considered to be
dependent on the distribution of LNP. Since the lipid composition of the luciferase RNA-
encapsulated LNP used in this study is identical to that of the application formulation of
BNT162b2, the results of this study are considered to indicate the distribution of
BNT162b2-encapsulated LNP.

PFIZER CONFIDENTIAL
Page 6

Masking area: under adjustment

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

5. Metabolism

Report Number: 01049 008, 01049 009, 01049f{ 010, 0104 020, 01049 021, 01049
022, PF-07302048 05 flo43725, Summary Table: 2.6.5.10A, 2.6.5.10B, 2.6.5.10C,
2.6.5.10 D

The in vitro metabolic stability of ALC-0315 and ALC-0159 was evaluated using liver
microsomes, liver S9 fractions, and hepatocytes from CD-1/ICR mice, Wistar Han or
Sprague Dawley rats, crab-eating macaques, and humans. ALC-0315 or ALC-0159 were
added to liver microsomes or liver S9 fractions (120 min incubation) or hepatocytes (240
min incubation) of each animal species, and the percentage of unchanged product after
incubation was determined. The results showed that ALC-0315 and ALC-0159 were
metabolically stable in both animal species and test systems, and the final percentage

of unchanged product was over 82%.

In addition, the metabolic pathways of ALC-0315 and ALC-0159 were evaluated in vitro
and in vivo. In these studies, in vitro metabolism was evaluated using blood, liver S9
fractions, and hepatocytes from CD-1 mice, Wistar Han rats, crab-eating macaques, and
humans. In addition, plasma, urine, feces, and liver samples collected in the rat PK study
were used to evaluate in vivo metabolism (Section M2.6.4.3). The test results showed
that the metabolism of both ALC-0315 and ALC-0159 was slow and metabolized by
hydrolysis of ester and amide bonds, respectively. Metabolism by hydrolysis, shown in

Figure 3 and Figure 4, was observed in all animal species evaluated.

PFIZER CONFIDENTIAL
Page 7

Masking area: under adjustment

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

Figure 3 The estimated metabolic pathway in organisms of ALC-0315
in various spices of animals

} 1O-~ yy eee eee

a O
'S OOCe
ALC-0815
in blood (mo, r) Poe iad ad i
in hepatocytes(mo, r, mk,h) oe
in liver s9 (mo, r, h) een cw
in plasma (r) cata Gediccls adiaastaaial in blood (mo, r)
‘ in liver s9 (mk)
o in plasma (r)
in blood (mo, r) cw m/z 528 in liver (r)
in hepatocytes(mo,r,mk,h) "Oy ~~~ ~
in liver s9 (mo, r, h) m/z 255
in plasma (r) ee anal in blood (mo, r)
aL in liver s9 (mk)
ee in plasma (r)
mz 200 in urine (r)
in feces (r)
in liver (r)
glucuronide
Ona Qn OH
u
M in urine (r)
Ou
m/z 466

H: Human, Mk: Monkey, Mo: Mouse, R: Rat

ALC-0315 is metabolized by undergoing two successive rounds of ester hydrolysis. These
two hydrolysis events produce first the monoester metabolite (m/z 528) and then the
double transesterification metabolite (m/z 290). The double transesterification
metabolite was further metabolized to a glucuronide conjugate (m/z 466), which was
detected only in urine in the rat PK study. It was also confirmed that the acidic products
of the two hydrolyses were both 6-hexyl decanoic acid (m/z 255).

PFIZER CONFIDENTIAL
Page 8

Masking area: under adjustment

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

Figure 4 Estimated metabolic pathway of ALC-0159 in various animal species

in blood (Mo, R)
in hepatocytes (Mo, R, Mk, H)
in liver S9 (Mo, R, Mk, H)

O
SOY
Yo ~ i “ N ———— i Me eh et is
° wWnWwnwanEOr—Oa™°.
| N,N-ditetradecyl amine

ALC-0159 mz 440

H: human, Mk: monkey, Mo: mouse, R: rat

The major metabolic pathway of ALC-0159 was the hydrolysis of amide bonds to form
N,N-ditetradecylamine (m/z 410). The metabolites were detected in mouse and rat blood,

mouse, rat, monkey, and human hepatocytes and liver S9 fractions.

6. Excretion

The concentrations of ALC-0315 and ALC-0159 were measured in urine and feces
collected over time in a PK study in which luciferase RNA-encapsulated LNPs were
administered intravenously to rats at a dose of 1 mg RNA/kg (Section M2.6.4.3). None of
the unchanged forms of ALC-0315 or ALC-0159 were detected in the urine. On the other
hand, unchanged forms of ALC-0315 and ALC-0159 were detected in the feces, and the
percentages per dose were about 1% and 50%, respectively. As shown in Figure 3,
metabolites of ALC-0315 were detected in urine.

7. Pharmacokinetic Drug Interactions

Pharmacokinetic drug interaction studies have not been conducted for this vaccine.

8. Other pharmacokinetic studies
No other pharmacokinetic studies have been conducted for this vaccine.

9. Discussion and Conclusion

In the rat PK study, plasma and liver ALC-0315 concentrations decreased to
approximately 1/7000 and 1/4 of the maximum concentrations, respectively, by 2 weeks
post-dose, and ALC-0159 concentrations decreased to approximately 1/8000 and 1/250
of the maximum concentrations, respectively. t% was similar in plasma and liver, 6-8 days
for LC-0315 and 2-3 days for ALC-0159. The t% was similar in plasma and liver, 6-8 days
for LC-0315 and 2-3 days for ALC-0159. Plasma t% values may represent the distribution
of each lipid in the tissues as LNP and its subsequent redistribution in the plasma during
the elimination process. The unchanged form of ALC-0315 was almost undetectable in
both urine and feces, but monoester metabolites, double transesterified metabolites, and
6-hexyl decanoate were detected in feces and plasma samples collected in the rat PK
study, and glucuronide conjugates of the double transesterified metabolite were
detected in urine. This metabolic process is thought to be the major mechanism of ALC-
0315 disappearance, but no quantitative data have been obtained to test this hypothesis.
On the other hand, about 50% of the dose of ALC-0159 was excreted in feces as
unchanged drug, and it was metabolized slowly by hydrolysis of amide bond in in vitro

metabolism experiments.

PFIZER CONFIDENTIAL
Page 9

Masking area: under adjustment
SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.4 Summary statement of the pharmacokinetic study

Since the biodistribution of the antigen encoded by BNT162b2 depends on the
distribution of LNP, we administered luciferase RNA-encapsulated LNP intramuscularly
to BALB/c mice and examined the biodistribution of alternative reporter proteins. The
results showed that luciferase was expressed at the site of administration, and was also
observed in the liver, although at a lower level. The expression of luciferase at the site of
administration was observed from 6 hours post-dose and disappeared by 9 days post-
dose. The expression of luciferase in the liver was observed from 6 hours post-dose and
disappeared by 48 hours post-dose. The distribution in the liver was considered to
indicate that the locally administered luciferase RNA-encapsulated LNP reached the
circulating blood and was taken up by the liver. When the radioactivity-conjugated form
of luciferase RNA-encapsulated LNP was administered intramuscularly to rats, the
highest radioactivity levels were observed at the site of administration. Outside of the
dose site, the highest radioactivity was detected in the liver, followed by the spleen,
adrenal glands, and ovaries, but the total radioactivity recovery relative to the dose in
these tissues was significantly lower than in the liver. This result is consistent with the
fact that luciferase expression was observed in the liver in the mouse biodistribution
study. No toxicity findings indicative of hepatic injury was observed in repeated-dose

toxicity studies in rats (see Section 2.6.6.3).

These non-clinical pharmacokinetic evaluations indicated that LNP reaching the
circulation was distributed in the liver, and the disappearance of ALC-0315 and ALC-

0159 was related to metabolism and fecal excretion, respectively.

10. Figures

Figures are shown in the text and in the summary tables.

References

1. World Health Organization. Annex 1. Guidelines on the non-clinical evaluation of
vaccines. In: WHO Technical Report Series No. 927, Geneva, Switzerland. World
Health Organization; 2005:31-63.

2. Guidelines on the non-clinical evaluation of vaccines for the prevention of infectious
diseases (Pharmaceutical and Food Safety Inspection Service No. 0527-1, May 27,
2010).

PFIZER CONFIDENTIAL
Page 10

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)
2.6.5 FSD) ReR ER D EES

2.6.5.1. PHARMACOKINETICS OVERVIEW Test Article: BNT162b2
Type of Study Test System Test item Method of Testing Facility Report Number
Administration
Single Dose Pharmacokinetics
Single Dose Rat (Wistar Han) modRNA encoding IV bolus Pfizer Inc* PF -07302048 0G 072424
Pharmacokinetics and luciferase
Excretion in Urine and Feces formulated in LNP
of ALC-0159 and ALC-0315 comparable to
BNT162b2
Distribution
In Vivo Distribution Mice BALB/c modRNA encoding IM Injection | R4g-0072
luciferase
formulated in LNP
comparable to
BNT162b2
In Vivo Distribution Rat (Wistar Han) modRNA encoding IM Injection a: 185350
luciferase
formulated in LNP
comparable to
BNT162b2 with
trace amounts of
[>H]-CHE as non-
diffusible label
Metabolism
In Vitro and In Vivo Metabolism
In Vitro Metabolic Stability Mouse (CD-1/ICR), rat ALC-0315 In vitro 01049008
of ALC-0315 in Liver (Sprague Dawley and
Microsomes Wistar Han), monkey
(Cynomolgus), and
human liver microsomes
In Vitro Metabolic Stability Mouse (CD-1/ICR), rat ALC-0315 In vitro 01049009
of ALC-0315 in Liver S9 (Sprague Dawley),
monkey (Cynomolgus),
and human S9 liver
fractions
PFIZER CONFIDENTIAL

Page 1

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.5 FSD) ReR ER D EES

2.6.5.1.

Type of Study

Test System

PHARMACOKINETICS OVERVIEW

Testing Facility

Test Article: BNT162b2

Report Number

In Vitro Metabolic Stability
of ALC-0315 in Hepatocytes

In Vitro Metabolic Stability

of ALC-0159 in Liver
Microsomes

In Vitro Metabolic Stability

of ALC-0159 in Liver S9

In Vitro Metabolic Stability
of ALC-0159 in Hepatocytes

Biotransformation of

ALC-0159 and ALC-0315 In

Vitro and In Vivo in Rats

Mouse (CD-1/ICR), rat
(Sprague Dawley and
Wistar Han), monkey

(Cynomolgus), and
human hepatocytes

Mouse (CD-1/ICR), rat
(Sprague Dawley and
Wistar Han), monkey

(Cynomolgus), and
human liver microsomes

Mouse (CD-1/ICR), rat

(Sprague Dawley),
monkey (Cynomolgus),
and human S9 fractions

Mouse (CD-1/ICR), rat
(Sprague Dawley and
Wistar Han), monkey

(Cynomolgus), and
human hepatocytes
In vitro:

CD-1 mouse, Wistar
Han rat, cynomolgus
monkey, and human
blood, liver S9 fractions
and hepatocytes
In vivo: male Wistar Han
rats

Pfizer Inc®

01049010

01049020

01049021

01049022

PF-07302048 OSM 043725

Test item Method of
Administration
ALC-0315 In vitro
ALC-0159 In vitro
ALC-0159 In vitro
ALC-0159 In vitro
ALC-0315 and In vitro or
ALC-0159 IV (in vivo in
rats)
PFIZER CONFIDENTIAL

Page 2

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FRB) ReRA ER D HEE

2.6.5.1. PHARMACOKINETICS OVERVIEW Test Article: BNT162b2

Type of Study Test System Test item Method of Testing Facility Report Number
Administration

ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), a proprietary polyethylene glycol-lipid included as an excipient in the LNP formulation
used in BNT162b2; ALC-0315 = (4-hydroxybutyl)azanediyl)bis(hexane-6, | -diyl)bis(2-hexyldecanoate), a proprietary aminolipid included as an excipient in the
LNP formulation used in BNT162b2; IM = Intramuscular; IV = Intravenous; LNP = lipid nanoparticles; S9 = Supernatant fraction obtained from liver
homogenate by centrifuging at 9000 g.

a. La Jolla, California.

b. , Germany.
Cc. , UK.
d. , China.

e. Groton, Connecticut.

PFIZER CONFIDENTIAL
Page 3

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FRB) ReRA ER D HEE

2.6.5.3. PHARMACOKINETICS: Test Article: modRNA encoding luciferase in LNP
PHARMACOKINETICS AFTER A SINGLE DOSE Report Number: PF-07302048_0g{l 072424
Species (Strain) Rat (Wistar Han)

Sex/Number of Animals Male/ 3 animals per timepoint®

Feeding Condition Fasted

Method of Administration IV

Dose modRNA (mg/kg) 1

Dose ALC-0159 (mg/kg) 1.96

Dose ALC-0315 (mg/kg) 15.3

Sample Matrix Plasma, liver, urine and feces

Sampling Time Points (h post dose): Predose, 0.1, 0.25, 0.5, 1, 3, 6, 24, 48, 96, 192, 336

Analyte ALC-0315 ALC-0159

PK Parameters: Mean? Mean?

AUCine (ugeh/mL)° 1030 99.2

AUCrast (ugeh/mL) 1020 98.6

Initial ty, (h)4 1.62 1.74

Terminal elimination ty, (h)° 139 d2eih

Estimated fraction of dose distributed to liver (%)! 59.5 20.3

Dose in Urine (%) NC& NC®

Dose in Feces (%)" 1.05 47.2

ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), a proprietary polyethylene glycol-lipid included as an excipient in the LNP formulation
used in BNT162b2; ALC-0315 = (4-hydroxybutyl)azanediyl)bis(hexane-6, | -diyl)bis(2-hexyldecanoate), a proprietary aminolipid included as an excipient in the
LNP formulation used in BNT162b2; AUCinr = Area under the plasma drug concentration-time curve from 0 to infinite time; AUCtast = Area under the plasma
drug concentration-time curve from 0 to the last quantifiable time point; BLQ = Below the limit of quantitation; LNP = Lipid nanoparticle;

modRNA = Nucleoside modified messenger RNA; PK = Pharmacokinetics; ty,= Half-life.

a. Non-serial sampling, 36 animals total.

b. Only mean PK parameters are reported due to non-serial sampling.

c. Calculated using the terminal log-linear phase (determined using 48, 96, 192, and 336 h for regression calculation).

d. In(2)/initial elimination rate constant (determined using 1, 3, and 6 h for regression calculation).

e. In(2)/terminal elimination rate constant (determined using 48, 96, 192, and 336 h for regression calculation).

f. Calculated as follows: highest mean amount in the liver (ug)/total mean dose (ug) of ALC-0315 or ALC-0159.

g. Not calculated due to BLQ data.

h. Fecal excretion, calculated as: (mean ug of analyte in feces/ mean pg of analyte administered) x 100

PFIZER CONFIDENTIAL
Page 4

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FRB) ReRA ER D HEE

2.6.5.55A. PHARMACOKINETICS: ORGAN
DISTRIBUTION

Test Article: modRNA encoding luciferase in LNP
Report Number: R§-0072

Species (Strain): Mice (BALB/c)
Sex/Number of Animals: Female/3 per group
Feeding Condition: Fed ad libitum
Vehicle/Formulation: Phosphate-buffered saline
Method of Administration: Intramuscular injection
Dose (mg/kg): 1 pg/hind leg in gastrocnemius muscle (2 pg total)
Number of Doses: 1
Detection: Bioluminescence measurement
Sampling Time (hour): 6, 24, 48, 72 hours; 6 and 9 days post-injection
Time point Total Mean Bioluminescence signal (photons/second) Mean Bioluminescence signal in
the liver (photons/second)
Buffer control modRNALuciferase in LNP modRNALuciferase in LNP
6 hours 1.28x10° 1.26x10° 4.94x107
24 hours 2.28x10° 7.31x108 2.4x10°
48 hours 1.40x10° 2.10108 Below detection*
72 hours 1.33x10° 7.87x107 Below detection*
6 days 1.62x10° 2.92x10° Below detection®
9 days 7.66104 5.09x10° Below detection®

LNP = Lipid nanoparticle; modRNA = Nucleoside modified messenger RNA.

a. At or below the background level of the buffer control.

PFIZER CONFIDENTIAL
Page 5

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FRB) ReRA ER D HEE

2.6.5.5B. PHARMACOKINETICS: ORGAN Test Article: [*>H]-Labelled LNP-mRNA formulation containing
DISTRIBUTION CONTINUED ALC-0315 and ALC-0159
Report Number: 185350
Species (Strain): Rat (Wistar Han)
Sex/Number of Animals: Male and female/3 animals/sex/timepoint (21 animals/sex total for the 50 ug dose)
Feeding Condition: Fed ad libitum
Method of Administration: Intramuscular injection
Dose: 50 wg [H]-08-A01-CO (lot # NC-0552-1)
Number of Doses: 1
Detection: Radioactivity quantitation using liquid scintillation counting
Sampling Time (hour): 0.25, 1, 2, 4, 8, 24, and 48 hours post-injection
Sample Mean total lipid concentration (ug lipid equivalent/g (or mL) % of administered dose (males and females combined)
(males and females combined)
0.25h lh 2h 4h 8h 24h 48h 0.25 h 1h 2h 4h 8h 24h 48h
Adipose tissue 0.057 0.100 0.126 0.128 0.093 0.084 0.181 -- -- -- -- -- -- --
Adrenal glands 0.271 1.48 2.72 2.89 6.80 13.8 18.2 0.001 0.007 0.010 0.015 0.035 0.066 0.106
Bladder 0.041 0.130 0.146 0.167 0.148 0.247 0.365 0.000 0.001 0.001 0.001 0.001 0.002 0.002
Bone (femur) 0.091 0.195 0.266 0.276 0.340 0.342 0.687 -- -- -- -- -- = --
Bone marrow 0.479 0.960 1.24 1.24 1.84 2.49 3.77 -- -- -- -- -- -- --
(femur)
Brain 0.045 0.100 0.138 0.115 0.073 0.069 0.068 0.007 0.013 0.020 0.016 0.011 0.010 0.009
Eyes 0.010 0.035 0.052 0.067 0.059 0.091 0.112 | 0.000 0.001 0.001 0.002 0.002 0.002 0.003
Heart 0.282 1.03 1.40 0.987 0.790 0.451 0.546 | 0.018 0.056 0.084 0.060 0.042 0.027 0.030
Injection site 128 394 311 338 213 195 165 19.9 52.6 31.6 28.4 21.9 29.1 24.6
Kidneys 0.391 1.16 2.05 0.924 0.590 0.426 0.425 0.050 0.124 0.211 0.109 0.075 0.054 0.057
Large intestine 0.013 0.048 0.093 0.287 ~=0.649 1.10 1.34 0.008 0.025 0.065 0.192 0.405 0.692 0.762
Liver 0.737 4.63 11.0 16.5 26.5 19.2 24.3 0.602 2.87 7.33 11.9 18.1 15.4 16.2
Lung 0.492 1.21 1.83 1.50 1.15 1.04 1.09 0.052 0.101 0.178 0.169 0.122 0.101 0.101

PFIZER CONFIDENTIAL
Page 6

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FRB) ReRA ER D HEE

2.6.5.5B. PHARMACOKINETICS: ORGAN
DISTRIBUTION CONTINUED

Test Article: [7>H]-Labelled LNP-mRNA formulation containing
ALC-0315 and ALC-0159
Report Number: 185350

Sample Total Lipid concentration (ug lipid equivalent/g [or mL]) % of Administered Dose (males and females combined)
(males and females combined)
0.25h lh 2h 4h 8h 24h 48h 0.25 h lh 2h 4h 8h 24h 48h
Lymph node 0.064 0.189 0.290 0.408 0.534 0.554 0.727 -- -- -- -- -- -- --
(mandibular)
Lymph node 0.050 0.146 0.530 0.489 0.689 0.985 1.37 -- -- -- -- -- -- --
(mesenteric)
Muscle 0.021 0.061 0.084 0.103 0.096 0.095 0.192 -- -- = -- -- -- --
Ovaries 0.104 1.34 1.64 2.34 3.09 5.24 12.3 0.001 0.009 0.008 0.016 0.025 0.037 0.095
(females)
Pancreas 0.081 0.207 ~=—0.414 0.380 0.294 0.358 0.599 | 0.003 0.007 0.014 0.015 0.015 0.011 0.019
Pituitary gland 0.339 0.645 0.868 0.854 0.405 0.478 0.694 | 0.000 0.001 0.001 0.001 0.000 0.000 0.001
Prostate 0.061 0.091 0.128 0.157. 0.150 0.183 0.170 | 0.001 0.001 0.002 0.003 0.003 0.004 0.003
males
ae 0.084 0.193 0.255 0.220 0.135 0.170 0.264 | 0.003 0.007 0.008 0.008 0.005 0.006 0.009
glands
Skin 0.013 0.208 0.159 0.145 0.119 0.157 0.253 -- -- -- -- -- -- --
Small intestine 0.030 0.221 0.476 0.879 1.28 1.30 1.47 0.024 0.130 0.319 0.543 0.776 0.906 0.835
Spinal cord 0.043 0.097 ~—0.169 0.250 0.106 0.085 0.112 | 0.001 0.002 0.002 0.003 0.001 0.001 0.001
Spleen 0.334 2.47 7.73 10.3 22.1 20.1 23.4 0.013 0.093 0.325 0.385 0.982 0.821 1.03
Stomach 0.017 0.065 0.115 0.144 0.268 0.152 0.215 | 0.006 0.019 0.034 0.030 0.040 0.037 0.039
Testes (males) 0.031 0.042 0.079 0.129 0.146 0.304 0.320 | 0.007 0.010 0.017 0.030 0.034 0.074 0.074
Thymus 0.088 0.243 =—-0.340 0.335 0.196 0.207 0.331 0.004 0.007 0.010 0.012 0.008 0.007 0.008
Thyroid 0.155 0.536 0.842 0.851 0.544 0.578 = 1.00 0.000 0.001 0.001 0.001 0.001 0.001 0.001
Uterus 0.043 0.203 0.305 0.140 0.287 0.289 0.456 | 0.002 0.011 0.015 0.008 0.016 0.018 0.022
(females)
Whole blood 1.97 4.37 5.40 3.05 1.31 0.909 0.420 -- -- -- -- -- -- --
Plasma 3.97 8.13 8.90 6.50 2.36 1.78 0.805 -- = -- -- -- -- --
Blood:Plasma 0.815 0.515 0.550 0.510 0.555 0.530 0.540 -- -- -- -- -- -- --
ratio*
PFIZER CONFIDENTIAL

Page 7

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FRB) ReRA ER D HEE

2.6.5.5B. PHARMACOKINETICS: ORGAN Test Article: [7H]-Labelled LNP-mRNA formulation containing

DISTRIBUTION CONTINUED ALC-0315 and ALC-0159
Report Number: 185350

-- = Not applicable, partial tissue taken; [>H]-08-A01-CO = An aqueous dispersion of LNPs, including ALC-0315, ALC-0159, distearoylphosphatidylcholine,
cholesterol, mRNA encoding luciferase and trace amounts of radiolabeled [Cholesteryl-1,2-3H(N)]-Cholesteryl Hexadecyl Ether, a nonexchangeable, non-
metabolizable lipid marker used to monitor the disposition of the LNPs; ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N--ditetradecylacetamide), a proprietary
polyethylene glycol-lipid included as an excipient in the LNP formulation used in BNT162b2; ALC-0315 = (4--hydroxybutyl)azanediyl)bis(hexane-6, | -
diyl)bis(2-hexyldecanoate), a proprietary aminolipid included as an excipient in the LNP formulation used in BNT162b2; LNP = Lipid nanoparticle;

mRNA = messenger RNA.

a. The mean male and female blood:plasma values were first calculated separately and this value represents the mean of the two values.

PFIZER CONFIDENTIAL
Page 8

SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048)

2.6.5 FRB) ReRA ER D HEE

2.6.5.9. PHARMACOKINETICS: METABOLISM IN VIVO,

RAT

Species (Strain):

Sex/ Number of animals

Method of Administration:

Dose (mg/kg):

Test System:

Analysis Method:
Biotransformation

N-dealkylation, oxidation
N-Dealkylation, oxidation
N-dealkylation, oxidation
N-Dealkylation, oxidation
N-dealkylation, hydrolysis, oxidation
Hydrolysis (acid)

Hydrolysis, hydroxylation
Bis-hydrolysis (amine)

Hydrolysis, glucuronidation
Bis-hydrolysis (amine), glucuronidation
Bis-hydrolysis (amine), glucuronidation
Hydrolysis (amine)

Hydrolysis (amine), Glucuronidation
Oxidation to acid

Oxidation to acid

Hydroxylation

Sulfation

Sulfation

Glucuronidation

Glucuronidation

m/z

102.0561?
104.0706°
130.0874?
132.1019°
145.0506
255.2330"
271.2279"
290.2690°
431.2650
464.2865
466.3011
528.4986
704.5307
778.6930
780.7076
782.7232
844.6706
846.6851
940.7458
942.7604

Tap TF ap TFT TFT PF TF TF BP PB

Test Article: modRNA encoding luciferase in LNP

Report Number: PF-07302048_0 | 043725
Rat (Wistar Han)
Male/ 36 animals total for plasma and liver, 3 animals for urine and feces
Intravenous
1
Plasma, Urine, Feces, Liver
Ultrahigh performance liquid chromatography/ mass spectrometry
Metabolites of ALC-0315 Detected
Plasma | Urine Feces | Liver

ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND

+ ND ND ND
ND ND ND ND

+ + + +
ND ND ND ND
ND ND ND ND
ND + ND ND

cb ND ND =H:
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND

Note: Both theoretical and observed metabolites are included.

m/z = mass to charge ratio; ND = Not detected; + = minor metabolite as assessed by ultraviolet detection.

a. Negative ion mode.
b. Positive ion mode.

PFIZER CONFIDENTIAL
Page 9

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FMB) RRR D HES

Test Article: ALC-0315
Report Numbers: 01049

2.6.5.10A. PHARMACOKINETICS: METABOLISM IN VITRO

Type of Study: Stability of ALC-0315 In Vitro
Study System: Liver Microsomes + NADPH S9 Fraction + NADPH, UDPGA, and Hepatocytes
alamethicin
ALC-0315 1 uM 1 uM 1 uM
Concentration:
Duration of 120 min 120 min 240 min
Incubation (min):
Analysis Method: Ultra-high performance liquid chromatography-tandem mass spectrometry
Incubation time Percent ALC-0315 remaining
(min) Liver Microsomes Liver S9 Fraction Hepatocytes
Mouse Rat Rat Monkey Human Mouse Rat(SD) Monkey Human | Mouse Rat Rat Monkey Human
(CD- (SD) (WH) (Cyno) (CD- (Cyno) (CD- (SD) (WH) = (Cyno)
1/ICR) 1/ICR) 1/ICR)
0 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
15 98.77 94.39 96.34 97.96 100.24 97.69 98.85 99.57 95.99 -- -- -- -- --
30 97.78 96.26 97.32 96.18 99.76 97.22 99.62 96.96 97.32 101.15 97.75 102.70 96.36 100.72
60 100.49 = 99.73 98.54 100.00 101.45 98.61 99.62 99.13 94.98 100.77 98.50 102.32 97.82 101.44
90 97.78 98.66 94.15 97.96 100.48 98.15 98.85 98.70 98.33 101.92 99.25 103.09 100.0 100.36
120 96.54 95.99 93.66 97.71 98.31 96.76 98.46 99.57 99.33 98.85 97.38 99.61 96.36 100.72
180 -- -- -- -- -- -- -- -- -- 101.15 98.88 103.47 95.64 98.92
240 -- - -- -- -- -- -- -- -- 99.62 101.12 100.00 93.82 99.64
ty (min) >120 >120 >120 >120 >120 >120 >120 >120 >120 >240 >240 >240 >240 >240

-- = Data not available; ALC-0315 = (4-hydroxybutyl)azanediyl)bis(hexane-6, | -diyl)bis(2-hexyldecanoate), a proprietary aminolipid included as an excipient in the lipid

nanoparticle formulation used in BNT162b2; Cyno = Cynomolgus; NADPH = Reduced form of nicotinamide adenine dinucleotide phosphate; NC = not calculated; SD = Sprague
Dawley; ty = half-life; WH = Wistar-Han; UDPGA= uridine-diphosphate-glucuronic acid trisodium salt.

PFIZER CONFIDENTIAL
Page 10

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FRB) ReRA ER D HEE

Test Article: ALC-0159
Report Numbers: 01049

2.6.5.10B. PHARMACOKINETICS: METABOLISM IN VITRO
CONTINUED

Type of Study: Stability of ALC-0159 In Vitro

Study System: Liver Microsomes + NADPH S9 Fraction + NADPH, UDPGA, and Hepatocytes
alamethicin

ALC-0159 1 uM 1 uM 1 uM

Concentration:

Duration of 120 min 120 min 240 min

Incubation (min):

Analysis Method: Ultra-high performance liquid chromatography-tandem mass spectrometry
Incubation time Percent ALC-0159 remaining
(min) Liver Microsomes Liver S9 Fraction Hepatocytes
Mouse Rat Rat Monkey Human Mouse Rat (SD) Monkey Human | Mouse Rat Rat Monkey Human
(CD- (SD) (WH) (Cyno) (CD-1/ICR) (Cyno) (CD- (SD) (WH) (Cyno)
1/ICR) 1/ICR)
0 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
15 82.27 101.24 =112.11 100.83 99.59 98.93 84.38 91.30 106.73 -- -- -- -- --
30 86.40 93.78 102.69 85.12 92.28 91.10 90.87 97.96 107.60 100.85 93.37 113.04 90.23 106.34
60 85.54 98.34 105.38 86.36 95.53 102.85 97.97 105.56 104.97 94.92 91.81 105.07 92.93 101.58
90 85.41 95.44 100.90 94.63 97.97 90.75 93.51 108.33 109.36 94.28 90.25 112.80 94.59 92.67
120 95.87 97.10 108.97 93.39 93.09 106.76 92.70 105.74 119.59 87.08 89.47 104.11 97.51 96.04
180 -- -- -- -- -- -- -- -- -- 94.92 93.96 102.90 89.81 93.66
240 -- -- -- -- -- -- -- -- -- 102.75 94.93 98.79 92.93 102.57
ty, (min) >120 >120 >120 >120 >120 >120 >120 >120 >120 >240 >240 >240 >240 >240

-- = Data not available; ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), a proprietary polyethylene glycol-lipid included as an excipient in the lipid
nanoparticle formulation used in BNT162b2; Cyno = Cynomolgus; NADPH = Reduced form of nicotinamide adenine dinucleotide phosphate; NC = not calculated; SD = Sprague
Dawley; WH = Wistar-Han; UDPGA= uridine-diphosphate-glucuronic acid trisodium salt.

PFIZER CONFIDENTIAL

Page 11

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FRB) ReRA ER D HEE

2.6.5.10C. PHARMACOKINETICS: METABOLISM Test Article: ALC-0315
IN VITRO CONTINUED Report Number: PF-07302048 0S 043725
Type of study Metabolism of ALC-0315 In Vitro
Study system Blood Hepatocytes Liver S9 Fraction
ALC-0315 concentration 10 uM 10 uM 10 uM
Duration of incubation 24h 4h 24h
Analysis Method: Ultrahigh performance liquid chromatography/ mass spectromet
Biotransformation Hepatocytes Liver S9 Fraction

Mouse | Rat | Monkey | Human | Mouse Rat Monkey | Human Mouse | Rat | Monkey Human
N-dealkylation, oxidation 102.0561# ND ND ND ND ND ND ND ND ND ND ND ND
N-Dealkylation, oxidation 104.0706" ND ND ND ND ND ND ND ND ND ND ND ND
N-dealkylation, oxidation 130.0874 ND ND ND ND ND ND ND ND ND ND ND ND
N-Dealkylation, oxidation 132.1019° ND ND ND ND ND ND ND ND ND ND ND ND
N-dealkylation, hydrolysis, oxidation 145.0506 ND ND ND ND ND ND ND ND ND ND ND ND
Hydrolysis (acid) 255.2330? ND ND ct + + | ND +
Hydrolysis, hydroxylation 271.2279 ND ND ND ND ND ND ND ND ND ND ND ND
Bis-hydrolysis (amine) 290.2690 ND ND ND ND ND ND ND ND + ND
Hydrolysis, glucuronidation 431.2650? ND ND ND ND ND ND ND ND ND ND ND ND
Bis-hydrolysis (amine), glucuronidation 464.2865? ND ND ND ND ND ND ND ND ND ND ND ND
Bis-hydrolysis (amine), glucuronidation 466.3011 ND ND ND ND ND ND ND ND ND ND ND ND
Hydrolysis (amine) 528.4986 ND + ND ND ND ND ND ND ND ND + ND
Hydrolysis (amine), glucuronidation 704.5307 ND ND ND ND ND ND ND ND ND ND ND ND
Oxidation to acid 778.6930 ND ND ND ND ND ND ND ND ND ND ND ND
Oxidation to acid 780.7076 ND ND ND ND ND ND ND ND ND ND ND ND
Hydroxylation 782.7232 ND ND ND ND ND ND ND ND ND ND ND ND
Sulfation 844.67067 ND ND ND ND ND ND ND ND ND ND ND ND
Sulfation 846.6851 ND ND ND ND ND ND ND ND ND ND ND ND
Glucuronidation 940.7458 ND ND ND ND ND ND ND ND ND ND ND ND
Glucuronidation 942.7604 ND ND ND ND ND ND ND ND ND ND ND ND
Note: Both theoretical and observed metabolites are included.
m/z = mass to charge ratio; ND = Not detected; + = metabolite present.
a. Negative ion mode.
b. Positive ion mode.

PFIZER CONFIDENTIAL

Page 12

SARS-CoV-2 mRNA Vaccine (BNT 162, PF-07302048)
2.6.5 FRB) ReRA ER D HEE

2.6.5.10D. PHARMACOKINETICS: METABOLISM Test Article: ALC-0159
IN VITRO CONTINUED Report Number: PF-07302048 05MM 043725
Type of study Metabolism of ALC-0159 In Vitro
Study system Blood Hepatocytes Liver S9 Fraction
ALC-0159 concentration 10 uM 10 uM 10 uM
Duration of incubation 24h 4h 24h
Analysis Method: Ultrahigh performance liquid chromatography/ mass spectromet
Biotransformation Hepatocytes Liver S9 Fraction
Mouse | Rat | Monkey | Human | Mouse Rat Monkey | Human | Mouse | Rat | Monkey | Human
O-Demethylation, O-dealkylation 107.0703 ND ND ND ND ND ND ND ND ND ND ND ND
O-Demethylation, O-dealkylation 151.0965 ND ND ND ND ND ND ND ND ND ND ND ND
O-Demethylation, O-dealkylation 195.1227 ND ND ND ND ND ND ND ND ND ND ND ND
Hydrolysis, N-Dealkylation 214.2529 ND ND ND ND ND ND ND ND ND ND ND ND
N-Dealkylation, oxidation 227.20178 ND ND ND ND ND ND ND ND ND ND ND ND
Hydrolysis (amine) 410.4720 + + ND ND + + +
N,N-Didealkylation 531.5849 ND ND ND ND ND ND ND ND ND ND ND ND
N-Dealkylation 580.6396 ND ND ND ND ND ND ND ND ND ND ND ND
O-Demethylation, oxidation 629.6853 ND ND ND ND ND ND ND ND ND ND ND ND
Hydroxylation 633.6931 ND ND ND ND ND ND ND ND ND ND ND ND
@-Hydroxylation, Oxidation 637.1880 ND ND ND ND ND ND ND ND ND ND ND ND
Hydrolysis (acid) 708.7721 ND ND ND ND ND ND ND ND ND ND ND ND
Note: Both theoretical and observed metabolites are included.
m/z = mass to charge ratio; ND = Not detected; + = metabolite present.
a. Negative ion mode.
b. Positive ion mode.
PFIZER CONFIDENTIAL

Page 13